T cell receptor–dependent S-acylation of ZAP-70 controls activation of T cells

Ritika Tewari,Bieerkehazhi Shayahati,Ying Fan,Askar M Akimzhanov

doi:10.1016/j.jbc.2021.100311

Abstract

ZAP-70 is a tyrosine kinase essential for T cell immune responses. Upon engagement of the T cell receptor (TCR), ZAP-70 is recruited to the specialized plasma membrane domains, becomes activated, and is released to phosphorylate its laterally segregated targets. A shift in ZAP-70 distribution at the plasma membrane is recognized as a critical step in TCR signal transduction and amplification. However, the molecular mechanism supporting stimulation-dependent plasma membrane compartmentalization of ZAP-70 remains poorly understood. In this study, we identified previously uncharacterized lipidation (S-acylation) of ZAP-70 using Acyl-Biotin Exchange assay, a technique that selectively captures S-acylated proteins. We found that this posttranslational modification of ZAP-70 is dispensable for its enzymatic activity. However, the lipidation-deficient mutant of ZAP-70 failed to propagate the TCR pathway suggesting that S-acylation is essential for ZAP-70 interaction with its protein substrates. The kinetics of ZAP-70 S-acylation were consistent with TCR signaling events indicating that agonist-induced S-acylation is a part of the signaling mechanism controlling T cell activation and function. Taken together, our results suggest that TCR-induced S-acylation of ZAP-70 can serve as a critical regulator of T cell-mediated immunity.

Highlights

Activation [3,4,5]
The newly exposed thiol groups were biotinylated and pulled down using streptavidin beads, and the captured proteins were analyzed by immunoblotting. This approach, in combination with a candidate-based screening of the proximal T cell receptor (TCR) signaling components, allowed us to detect previously uncharacterized S-acylation of several key TCR signaling proteins, including adaptor protein GRB2, phospholipase PLC-γ1, and tyrosine kinase ZAP-70 in both Jurkat cell line and primary human CD4+ T cells (Fig. 1, A and B). These findings suggested that this posttranslational modification is more widespread than initially thought and prompted us to investigate a possible role of S-acylation in the regulation of the ZAP-70 signaling function
We identified C564 as the site of a posttranslational modification known as protein S-acylation

Summary

Results

We first identified ZAP-70 as an S-acylated protein in our initial experiments aimed at the detection of novel lipidated proteins in the proximal TCR signaling pathway. The newly exposed thiol groups were biotinylated and pulled down using streptavidin beads, and the captured proteins were analyzed by immunoblotting This approach, in combination with a candidate-based screening of the proximal TCR signaling components, allowed us to detect previously uncharacterized S-acylation of several key TCR signaling proteins, including adaptor protein GRB2, phospholipase PLC-γ1, and tyrosine kinase ZAP-70 in both Jurkat cell line and primary human CD4+ T cells (Fig. 1, A and B). These findings suggested that this posttranslational modification is more widespread than initially thought and prompted us to investigate a possible role of S-acylation in the regulation of the ZAP-70 signaling function. Negative controls showed no detectable phosphorylation of SLP-76, we found that the C564R mutation did not cause any apparent loss of ZAP-70 kinase activity as evident from robust

75 ZAP-70

Discussion

Experimental procedures